Microscopy, Culturing, and Counting (Lab) Flashcards
What is resolution?
The ability to distinguish separate objects as actually being separate
How do you improve resolution?
Use a smaller wavelength
Are smaller or larger resolutions better?
Smaller
Why do we use oil immersion?
Causes the light to travel in a straight path instead of being refracted by the air, which causes more light to enter the objective
Why do we use Koehler illumination?
Provides the best possible image by setting up the light path correctly
What are the 2 purposes of aseptic technique?
To protect yourself and the environment from your experiment
To protect your experiment from yourself and the environment
What are 5 ways to do aseptic technique?
- Wash your hands
- Ethanol the bench
- Keep lids on
- Don’t put lids down on contaminated surfaces
- Flame culture tubes
Why do we streak plates for single colonies?
Allows us to purify colonies to get a culture of genetically identical cells
What are 2 ways to sterilize stuff?
Dry heat: flaming with bunsen burners
Moist heat: autoclave - high pressure and heat
Which method of sterilization can kill endospores? Dry heat or moist heat?
Moist heat (the autoclave)
Are plates labelled on the lid or the bottom?
Bottom. Labelling on the lid would become difficult if the lids accidentally became switched
Why do we do serial dilutions?
Determine the appropriate dilution that gives between 30-300 colonies when plated, then using that to calculate the original cell density
Why would we use a pure liquid culture over a pure culture on an agar plate?
Much easier to get DNA or proteins out of a cell suspension than a plate
What are the 6 different types of medium?
- Defined
- Minimal
- Complex
- Enriched
- Selective
- Differential
What is defined medium?
We know the exact nutrient composition of both organic and inorganic compounds
What is minimal medium?
A specific type of defined medium where only prototrophic cells can grow. Contains inorganic salts, ions, and a carbon source. Everything else needs to be synthesized de novo by the cells
What is complex medium?
Highly nutritious medium full of organic and inorganic energy sources made from a digest of plant, animal, or microbial products (Casein, soy, yeast, beef). Don’t know the exact nutrient composition
What is enriched medium?
Complex medium + a few other things like blood or serum that supports the growth of fastidious microorganisms
What is selective medium?
Medium that only allows the growth of certain microorganisms
What is differential medium?
Complex medium containing some sort of dye that will allow different properties to be distinguished
Why is all growth medium somewhat selective?
No medium will support the growth of every single bacterial species
What are endospores?
A differentiated dormant cell type produced by some species that are resistant to desiccation, radiation, heat
How do you break the dormancy on an endospore?
High heat treatment
What are total cell counts?
All cells are counted, whether alive, dead, or dormant
What are viable cell counts?
Only cells that are alive and capable of forming colonies are counted
What are 3 direct ways to get a total cell count?
- Microscopy to look at the cells
- Hemocytometer without methylene blue
- Petroff-Hausser counter
What are 3 indirect ways to get a total cell count?
- Turbidity measurements with OD
- Dry weight
- Measurements of cell components (ex. DNA)
What is the direct way to get a viable cell count?
Hemocytometer with methylene blue
What are 2 indirect ways to get a viable cell count?
- Serial dilution and plate count
2. Measurement of incorporation of an indicator molecule
How do you calculate the original viable cell concentration from a plate count? What are the units?
Average # of colonies on a plate/dilution factor x volume of dilution plated
Units are CFU/ml
Why can plate counts sometimes be inaccurate?
One colony didn’t necessarily stem from 1 cell. Lots of bacteria show clumping, and it is impossible to distinguish different colonies
Why does the methylene blue dye stain alive and dead cells differently?
Dead cells have compromised membranes, so the dye can get in
How do you get a viable cell count from a hemocytometer?
of alive cells - # of dead cells/# of total cells
